PEG-maleimide labelling

PEG-maleimide labelling can be used to determine how many cysteine residues of a protein are exposed to the surface and can be expected to react with maleimide conjugated dyes. The advantage of labelling with PEG is that it causes a large change in the apparent molecular mass of the protein which can be detected by SDS-PAGE.

• Make a stock of PEG-maleimide in DMSO (a 1mM stock is easily possible)

• Ensure that there is no reducing agent in your protein buffer (desalt if necessary)

• Mix protein and PEG together in molar ratio of 1:10. Ensure that the final [DMSO] is low (eg 1%).

• Take aliquots from the reaction throughout a timecourse and quench the reaction in each aliquot with DTT. A first-guess timecourse should run from 30 s to 15 min

• Run a gel of the aliquots - a ladder should appear as the bands shift. Running a timecourse should enable easy quantification of the number of sites available as intermediate steps are visible on the ladder.